Identification of the redox partners of ERdj5/JPDI,a PDI family member,from an animal tissue

ERdj5 (also known as JPDI) is a member of PDI family conserved in higher eukaryotes. This protein possesses an N-terminal J domain and C-terminal four thioredoxin domains each having a redox active site motif. Despite the insights obtained at the cellular level on ERdj5, the role of this protein in vivo is still unclear. Here, we present a simple method to purify and identify the disulfide-linked complexes of this protein efficiently from a mouse tissue. By combining acid quenching and thiol-alkylation, we identified a number of potential redox partners of ERdj5 from the mouse epididymis. Further, we show that ERdj5 indeed interacted with two of the identified proteins via formation of intermolecular disulfide bond. Thus, this approach enabled us to detect and identify redox partners of a PDI family member from an animal tissue.     

Minor Constituents of Japanese Ho-Leaf Oil

The main component of Japanese Ho-leaf oil has been shown to be (?)-linalool (80~90%), and the following twenty minor constituents newly have been identified; methyl vinyl ketone, methyl isobutyl ketone, mesityl oxide, β-pinene, myrcene, (+)-limonene, cis- and trans-ocimene, n-hexanol, cis-3-hexenol, cis- and trans-linalool oxide, (?)-1-terpinen-4-ol, (+)-cis and (+)-trans-2,6,6-trimethyl-2-vinyl-5-hydroxytetrahydropyran, citronellol, nerol, (+)-β-selinene, (+)-tagetonol and (?)-trans-hotrienol. (+)-Tagetonol and (?)-trans-hotrienol have been demonstrated to be (+)-3,7-dimethyl-3-hydroxy-1-octen-5-one (III) and (3R)-(?)-trans-3,7-dimethyl-3-hydroxy-1,5,7-octatriene (IX), respectively.     

Calcification process dynamics in coral primary polyps as observed using a calcein incubation method

Calcification processes are largely unknown in scleractinian corals. In this study, live confocal imaging was used to elucidate the spatiotemporal dynamics of the calcification process in aposymbiotic primary polyps of the coral species Acropora digitifera. The fluorophore calcein was used as a calcium deposition marker and a visible indicator of extracellular fluid distribution at the tissue-skeleton interface (subcalicoblastic medium, SCM) in primary polyp tissues. Under continuous incubation in calcein-containing seawater, initial crystallization and skeletal growth were visualized among the calicoblastic cells in live primary polyp tissues. Additionally, the distribution of calcein-stained SCM and contraction movements of the pockets of SCM were captured at intervals of a few minutes. Our experimental system provided several new insights into coral calcification, particularly as a first step in monitoring the relationship between cellular dynamics and calcification in vivo. Our study suggests that coral calcification initiates at intercellular spaces, a finding that may contribute to the general understanding of coral calcification processes.     

Newly developed selective immunoinactivation assay revealed reduction in adipose triglyceride lipase activity in peripheral leucocytes from patients with idiopathic triglyceride deposit cardiomyovasculopathy

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare and newly identified disease among patients requiring cardiac transplantation. TGCV is characterized by cardiomyocyte steatosis and triglyceride (TG)-deposit atherosclerosis, resulting from the abnormal intracellular metabolism of TG. TGCV is classified into primary and idiopathic types. Primary TGCV carries ultra-rare genetic mutations in the adipose triglyceride lipase (ATGL), a rate-liming enzyme that hydrolyzes intracellular TG in adipose and non-adipose tissues. Idiopathic TGCV, first identified among autopsied individuals with diabetes mellitus (DM) with severe heart diseases, shows no ATGL mutations and its causes and underlying mechanisms are still unknown. TGCV is difficult to diagnose in daily clinics, thereby demanding feasible diagnostic procedures. We aimed to develop an assay to measure ATGL activity using peripheral leucocytes. Human his6-ATGL was expressed in COS1 cells, purified to homogeneity, and used to raise a polyclonal antibody neutralizing TG-hydrolyzing activity of ATGL. We developed a selective immunoinactivation assay (SIIA) for the quantitation of ATGL activity in cell lysates of leucocytes by the antibody neutralizing ATGL activities. ATGL activity was measured in 13 idiopathic TGCV patients, with two patients with primary TGCV as the negative control. Healthy (non-DM) and DM controls without heart diseases were also subjected. The developed SIIA assay revealed significant reduction in ATGL activity in leucocytes from patients with idiopathic TGCV who did not carry ATGL mutations as compared with non-DM and DM controls. Thus, ATGL in leucocytes may be an important biomarker for the diagnosis of TGCV and our assay may provide insights into pathophysiology and elucidate the underlying mechanism of TGCV and related disorders.     

Isolation and Biological Activity of a Peptidal Antibiotic P168

The structure of the xyloglucan synthesised in vitro by the particulate fraction of suspension-cultured soybean (Glycine max) cells from UDP-glucose and UDP-xylose is mainly composed of two kinds of oligosaccharide-building blocks, a heptasaccharide unit and a pentassaccharide unit [T. Hayashi and K. Matsuda, J. Biol Chem., 256, 11117 (1981)]. The synthesis of the pentasaccharide unit is probably the first step in the construction of oligosaccharide building blocks to elongate the ²-1,4-glucan backbone. This enzymatically synthesized xyloglucan was shown to have the same molecular size (M_w, 180,000) as the xyloglucan prepared from soybean cell walls by gel filtration on a Sepharose CL-6B column, and the same building blocks distributed among each fraction. A pulse-chase experiment indicated that the pentasaccharide unit was converted into the heptasaccharide unit. The conversion was regulated by the concentration of UDP-xylose.     

Ligand Binding and Crystal Structures of the Substrate-Binding Domain of the ABC Transporter OpuA

Background

The ABC transporter OpuA from Lactococcus lactis transports glycine betaine upon activation by threshold values of ionic strength. In this study, the ligand binding characteristics of purified OpuA in a detergent-solubilized state and of its substrate-binding domain produced as soluble protein (OpuAC) was characterized.

Principal Findings

The binding of glycine betaine to purified OpuA and OpuAC (K_D = 4–6 µM) did not show any salt dependence or cooperative effects, in contrast to the transport activity. OpuAC is highly specific for glycine betaine and the related proline betaine. Other compatible solutes like proline and carnitine bound with affinities that were 3 to 4 orders of magnitude lower. The low affinity substrates were not noticeably transported by membrane-reconstituted OpuA. OpuAC was crystallized in an open (1.9 Å) and closed-liganded (2.3 Å) conformation. The binding pocket is formed by three tryptophans (Trp-prism) coordinating the quaternary ammonium group of glycine betaine in the closed-liganded structure. Even though the binding site of OpuAC is identical to that of its B. subtilis homolog, the affinity for glycine betaine is 4-fold higher.

Conclusions

Ionic strength did not affect substrate binding to OpuA, indicating that regulation of transport is not at the level of substrate binding, but rather at the level of translocation. The overlap between the crystal structures of OpuAC from L.lactis and B.subtilis, comprising the classical Trp-prism, show that the differences observed in the binding affinities originate from outside of the ligand binding site.     

OBSERVATIONS ON THE LIFE HISTORY OF PAPENFUSSIELLA CALLITRICHA (PHAEOPHYCEAE,CHORDARIALES) IN CULTURE1

Papenfussiella callitricha (Rosenv.) Kylin from eastern Canada was studied in culture. Zoids from unilocular sporangia develop into microscopic, filamentous, dioecious gametophytes which produce isogametes in filament cells and few-chambered plurilocular gametangia. Unfused gametes germinate to reproduce the gametophytes. Fusion takes place between a settled (“female”) and a motile (“male”) gamete. The zygote gives rise to a filamentous plethysmothallus that reproduces asexually by zoids formed in thallus cells and in few-chambered plurilocular zoidangia. Erect macrothalli are produced on the plethysmothallus, beginning with the formation of upright filaments. Later on, these filaments become the terminal assimilators of the macrothalli. Further assimilatory filaments, rhizoids, and unilocular sporangia are produced in a branching region at the base of the terminal assimilator. Zoids from unilocular sporangia formed in culture germinate to reestablish the gametophyte phase. Chromosome counts yielded n = 19 ± 3 for the gametophytes, and 32 ± 6 for the sporophyte, both plethysmothallus and macrothallus.     

ADVENTIVE EMBRYOGENESIS IN CITRUS I. THE OCCURRENCE OF ADVENTIVE EMBRYOS WITHOUT POLLINATION OR FERTILIZATION

Anatomical studies of unfertilized undeveloped seeds from open- and control-pollinated fruits of ten facultative apomictic Citrus cultivars were carried out with the aid of light and epifluorescence microscopes. With or without pollination, adventive embryos autonomously developed at all positions in the nucellus in all cultivars. The adventive embryos initiated at the chalazal end of the nucellus were more vigorous than those initiated at the micropylar end. Because of the lack of endosperm and poor seed development, however, all adventive embryos within the unfertilized seeds terminated their development at the globular or early cotyledonary stages and were unable to germinate under natural conditions. The capability of unfertilized seeds to develop varied from species to species. Growth of the adventive embryos was dependent on nucellus size, but the growth rate of adventive embryos relative to nucellus size was different in different species. Neither pollination, fertilization nor subsequent zygote and endosperm development further stimulated adventive embryo initiation. Conversely, pollination and subsequent fertilization of other seeds in the same fruit slightly, but significantly, suppressed adventive embryo growth in the unfertilized seeds. These facts concerning adventive embryogenesis in unfertilized seeds indicate that neither pollination nor fertilization is essential for in vivo adventive embryogenesis and that normal endosperm is necessary for perfect development of adventive embryos initiated only in the micropylar half of the nucellus.